In biology, biotechnology, chemistry and related areas such as bio-chemistry, yeasting factories, breweries, . . . , practical use is made of reactors, containers or incubators comprising a fluid medium, or samples which contain a fluid medium, into which certain processes occur, whereby the environmental parameters are under control. Examples are cell diagnostics and research laboratories where cell processes are to be monitored or observed, breweries such as beer breweries, where yeasting processes may have to be closely monitored, etc. Other examples are fermentators or fermentation reactors, water supply systems, plumbing, sewer systems, water canalizations, water quality improving and/or inspection installations or water purification plants, etc. where the objects in suspension are to be monitored or analyzed. To analyze and/or monitor the state and/or processes in the reactor, one has a choice between bringing the analysis apparatus to the reactor or taking samples from the reactor to the analysis apparatus. In the former case, typical problems are e.g. that the analysis apparatus needs to be resistant to the specific environment in the reactor, that the apparatus, when reused with another reactor, does not contaminate this other reactor, that the analysis apparatus is very expensive, that the apparatus is not accurate enough, etc.; in the latter case, a typical problem is the often time-consuming and/or labor-intensive gathering and preparing of samples for further observation or analysis. In such cases, it may be impossible to accurately monitor the state and/or processes of the reactor, as the time delay between the gathering of a sample and the analysis results may become too big.
Patent application US 2010/0315501 A1 discloses an electronic imaging flow-microscope for remote environmental sensing, bioreactor process monitoring, and optical microscopic tomography applications. Hereby, a fluid conduit has a port on each end of a thin flat transparent fluid transport region. A planar illumination surface contacts one flat side of the transparent fluid transport region and a planar image sensing surface contacts the other flat side. Light from the illumination surface travels through the transparent fluid transport region to the planar image sensing surface, producing a light field affected by the fluid and objects present. The planar image sensing surface creates electrical image signals responsive to the light field. The planar illumination surface can be light emitting elements such as LEDs, OLEDs, or OLET, whose illumination can be sequenced in an image formation process. The flow microscope can further comprise flow-restricting valves, pumps, energy harvesting arrangements, and power management.
However, traditional flow microscopes do not always provide enough information on the objects suspended in a flow. In some applications, three-dimensional data is to be acquired from these objects. Therefore, digital holographic imaging techniques may be applied.
Holography is a three-dimensional (3D) imaging technique that makes use of the interference between a reference wave and a wave emanating from the sample called object wave. The purpose of this interference is to record the phase of the object wave, which is related to the 3D character of the sample. With digital holographic imaging (DHI), real-time observations can be achieved by using a charged coupled device (CCD) camera as recording device and by performing a numerical reconstruction of the hologram. This idea has been proposed for the first time over 30 years ago by J. W. Goodmann, R. W. Lawrence, in “Digital image formation from electronically detected holograms,” Appl. Phys. Lett, Vol. 11, 1967. As a result of technological progresses achieved in the fields of digital image acquisition and processing, this numerical or digital approach of holography has considerably extended the fields of its potential applications and different types of DHI-inspired imaging systems have been developed during the last years.
DHI techniques can be classified in two main categories: in-line techniques characterized by the fact that the reference and object waves have similar propagation directions, and off-axis techniques for which the two interfering waves propagates along different direction. The procedure for hologram formation in in-line digital holography is similar to the procedure used for phase measurements with so-called phase-shifting interferometric techniques. Hologram formation with in-line techniques requires the acquisition of several images, at least three, that must be recorded during a modulation of the reference phase. Off-axis techniques, are more simple from the experimental point of view since they require a single hologram acquisition without modulation of the phase of the reference wave. In-line techniques however present the advantage that the reconstructed images are free of twin images and zero order of diffraction. Among off-axis techniques, we can distinguish methods based on Fourier-transform holography, and methods based on a so-called Fresnel holography. With Fourier-transform methods the reference wave must be a spherical wave of precisely controlled curvature and image reconstruction is basically performed by Fourier transformation of the hologram. With Fresnel-holography based techniques, the reconstruction procedure is more sophisticated but more flexibility is offered to build experimental installations.
Among recent publications presenting developments or applications of DHI-inspired techniques, we can mention the following works. A study of some general performances of an in-line technique is presented in “Image formation in phase-shifting digital holography and application to microscopy”, 1. Yamaguchi et al., Applied Optics, Vol. 40, No. 34, 2001, pp. 6177-6186. In “Fourier-transform holographic microscope”, Applied Optics, Vol. 31, 1992, pp. 4973-4978, W. S. Haddad et al describe the general principle of Fourier-transform DHI.
Examples of applications of the Fresnel-based approach can be found in “Direct recording of holograms by a CCD target and numerical reconstruction”, U. Schnars and W. Juptner, Applied Optics, Vol. 33, 1994, pp. 179-181, and in “Performances of endoscopic holography with a multicore optical fiber”, O. Coquoz et al., Applied Optics, Vol. 34, 1995, pp. 7186-7193.
A key element of a DHI method is the numerical method used for hologram reconstruction. An original reconstruction procedure, which allows for reconstructing simultaneously the amplitude and the phase of the object wave, on the basis of a single off-axis hologram acquisition, has been developed by Cuche et al. and is presented in U.S. Pat. No. 6,262,218, and in WO 00/20929. Different applications and implementations of this technique are presented in “Digital holography for quantitative phase-contrast imaging”, Optics Letters, Vol. 24, 1999, pp. 291-293, in “Simultaneous amplitude-contrast and quantitative phase-contrast microscopy by numerical reconstruction of Fresnel off-axis holograms”, Applied Optics, Vol. 38, 1999, pp. 6994-7001, in “Spatial Filtering for Zero-Order and Twin-Image Elimination in Digital Off-Axis Holography”, Applied Optics, Vol. 38 No. 34, 1999, in “Aperture apodization using cubic spline interpolation: Application in digital holographic microscopy”, Optics Communications, Vol. 182, 2000, pp. 59-69, and in “Polarization Imaging by Use of Digital Holography”, T. Colomb et al., Applied Optics, Vol. 38, No 34, 1999.
DHI method presents interesting possibilities of applications in cell biology. Indeed a living cell behaves optically as a phase object, i.e. a transparent sample whose constituents can be optically probed on the basis of the phase shift they induce on the light crossing them.
The phase-shifting behavior of transparent sample is well known, and for a long time as it constitutes the mechanism of image formation in phase-contrast (PhC) and Nomarski (DIC) microscopy. Even though these two techniques are widely used in biological microscopy, and well suited as contrasting methods, they cannot be used for precise quantitative phase measurements. The DHI method instead, is reminiscent of classical interferometry, which is the most commonly used technique for phase measurements. However, whereas interferometric techniques are widely used in metrology, only few biological applications have been reported, by R. Barer and S. Joseph, in “Refractometry of living cells”, Quarterly Journal of Microscopical Science, Vol. 95, 1954, pp. 399-423, by R. Barer in “Refractometry and interferometry of living cells”, Journal of the Optical Society of America, Vol. 47, 1957, pp. 545-556, by A. J. Coble et al. in “Microscope interferometry of necturus gallblader epithelium”, Josiah Macy Jr. Fundation, New York, 1982, p. 270-303, by K. C. Svoboda et al. in “Direct observation of kinesin stepping by optical trapping interferometry”, Nature, Vol. 365, 1993, by J. Farinas and A. S. Verkman, in “Cell volume plasma membrane osmotic water permeability in epithelial cell layers measured by interferometry”, Biophysical Journal, Vol. 71, 1996, pp. 3511-3522, by G. A. Dunn and D. Zicha in “Dynamics of fibroblast spreading”, Journal of Cell Science, Vol. 108, 1995, pp. 1239-1249.
For biological applications, as well as for material science or metrology applications, DHI methods offer a novel alternative to classical interferometry with similar performances but simplified experimental procedures. The main advantage originates from the fact that complex and costly experimental optical devices can be handled by digital processing methods. For example, as described by E. Cuche et al. in “Simultaneous amplitude-contrast and quantitative phase-contrast microscopy by numerical reconstruction of Fresnel off-axis holograms”, Applied Optics, Vol. 38, 1999, pp. 6994-7001, the wave front deformations appearing when a microscope objective is introduced along the path of the object wave can be compensated using a digital procedure. This particular feature opens attractive possibilities in the fields of microscopy. In addition DHI techniques performs faster than interferometric techniques, and provides more information about the sample, in particular, the amplitude and the phase of the object wave can be obtained simultaneously on the basis of a single hologram acquisition.
DHI methods have been applied to static imaging of biological cells, without phase reconstruction by K. Boyer et al. in “Biomedical three-dimensional holographic micro-imaging at visible, ultraviolet and X-ray wavelength”, Nature Medicine, Vol. 2, 1996, pp. 939-941, and by F. Dubois et al. in “Improved three-dimensional imaging with a digital holography microscope with a source of partial spatial coherence”, Applied Optics, Vol. 38, 1999, pp. 7085-7094. DHI of cells using a phase measurement modality requiring several image acquisitions has been reported by G. Indebetouw and P. Klysubun in “Saptiotemporal digital microholography”, Journal of the Optical Society of America A, Vol. 18, 2001, pp. 319-325.
With DHI, image acquisition can be performed at video-rate, and even faster using appropriate image acquisition systems, for experimental periods of up to several hours. Due to its interferometric nature, DHI has a high axial resolution (nanometer scale), which allows for observing subtle and minute modifications of sample shape, opening a wide field of applications in both life and material sciences. With the event of video-rate image acquisition by DHI, it has become possible to use DHI with a flow microscope, even at high flow rates.
WO2003048868 discloses an apparatus and a method for performing digital holographic imaging of a sample which includes a holographic creation unit, a holographic reconstruction unit, a processing unit, and a sample unit. The sample unit includes a container that contains a medium in which a sample is located.
U.S. Pat. No. 7,463,366 discloses a method and device for obtaining a sample with three-dimensional microscopy, in particular a thick biological sample and the fluorescence field emitted by the sample. One embodiment includes obtaining interferometric signals of a specimen, obtaining fluorescence signals emanating from the specimen, recording these signals, and processing these signals so as to reconstruct three-dimensional images of the specimen and of the field of fluorescence emitted by the specimen at a given time. Another embodiment includes a digital holography microscope, a fluorescence excitation source illuminating a specimen, where the microscope and the fluorescence excitation source cooperate to obtain interferometric signals of the specimen and obtain fluorescence signals emanating from the specimen, means for recording the interferometric signals and fluorescence signals, and means for processing the interferometric signals and the fluorescence signals so as to reconstruct three-dimensional images of the specimen and of the field of fluorescence emitted by the specimen at a given time.
Patent application WO2004102111 discloses a compact microscope able to work in digital holography for obtaining high quality 3D images of samples, including fluorescent samples and relatively thick samples such as biological samples, said microscope comprising illumination means at least partially spatially coherent for illuminating a sample to be studied and a differential interferometer for generating interfering beams from said sample on the sensor of an electronic imaging device, said interferometer comprising namely tilting means for tilting by a defined angle one the interfering beams relatively to the other, said tilting resulting into a defined shift of said interfering beam on the sensor of the electronic imaging device, said shift being smaller than spatial coherence width of each beam, said microscope being able to be quasi totally preadjusted independently from the samples so that minimum additional adjustments are required for obtaining reliable 3D images of samples.
However, the above mentioned prior art DHI techniques do not disclose how one can obtain data from an extensive sample of objects suspended in a fluid, nor the possibility of obtaining such data within a relatively short period. More in particular, most prior art DHI techniques focus on the imaging of small samples contained within a small specimen, whereby the accuracy and 3D imaging of DHI is being exploited, rather than its high rate of obtaining 3D information.
The problems in the prior art are multiple. The data acquired with the analysis apparatus of the prior may not be accurate enough, it may not be obtained quickly enough, the apparatus may be too expensive, it may only give two-dimensional and/or analogue images whereby three-dimensional information is obtained only after e.g. making a set of 2D images, digitalization and performing a CT-processing step. More in particular, DHMs may provide images and/or directly digitalized information about samples which is superior to other imaging or analysis techniques, but can be rather expensive. Furthermore, the gathered sample may need to be processed before analysis, which can be a time-consuming and labor-intensive procedure. Contamination may be an issue when the same apparatus is used to monitor or analyze different reactors, or the same reactor at different positions of times. Prior art techniques may not always provide the possibility of returning the sample to the reactor or to another reactor, or the possibility of real-time monitoring and providing timely feedback for adapting the reactor's environmental parameters.
There remains a need in the art for an improved system for the monitoring and/or analysis of one or more reactors and/or incubators comprising a fluid medium or comprising sample containing a fluid medium, in particular biological or biochemical cultures of organisms such as cells, bacteria, yeasts, micro-organisms, nematodes or any combination thereof, preferably in a liquid.
The present invention aims to resolve at least some of the problems mentioned above.
The invention thereto aims to provide an improved system for the monitoring and/or analysis of one or more reactors and/or incubators comprising a digital holographic microscope (DHM) and one or more fluidic systems which are capable of guiding a sample of the contents of a reactor with a fluid medium to the DHM for analysis and preferably back to the reactor. As such, one DHM can serve to analyze or monitor multiple reactors, and/or one reactor at different positions, e.g. at different heights, or at different times. The fluidic systems may be arranged such that contamination is avoided and replacement is easy.